Ten SEC gene products (SEC1, 2, 3, 4, 5, 6, 8, 9, 10, and 15) are required at the final stage of the yeast secretory pathway, but are not required for earlier stages. We have cloned nine and sequenced eight of these genes. Extensive genetic interactions suggest that the gene products work in a concerted fashion, possibly under regulation by Sec4. Sec4 is a GTP-binding protein of the rab branch of the ras superfamily. Sec4 undergoes a cycle of GTP binding, hydrolysis and exchange that may be coupled to a cycle of localization in which Sec4 is attached to secretory vesicles, the vesicles fuse with the plasma membrane and Sec4 recycles through the cytoplasm to bind to a new round of vesicles. While several of the accessory protein that regulate this cycle have been identified, a key remaining question is the nature of the effector pathway regulated by Sec4 that leads to exocytosis. Six aims are proposed: Sec8, Secl5, Sec6 and possibly additional Sec proteins form a 19.5 S particle that can peripherally associated with the plasma membrane and may constitute the immediate downstream effector of Sec4. The structure and function of the Sec8/Sec 15 complex will be probed. We will purify the complex to homogeneity and define all of its components. We will analyze the complex for the presence of all of the SEC gene products. Genes encoding any unidentified components will be cloned and their role in secretion addressed genetically. The interaction of the complex with Sec4 will be analyzed by both biochemical and genetic approaches. The association of the complex with the plasma membrane will be probed by an in vitro attachment assay. The role of ATP hydrolysis in the membrane attachment process will be defined. The membrane receptor and soluble co-factors will be identified. We will determine if the amino terminal region of Sec2, predicted to form a coiled-coil domain, mediates homo-dimer or hetero-dimer formation. We will use a genetic approach to define components that interact with the essential domain adjacent to the coiled-coil domain. SEC1 will be mutagenized to determine if an allele can be isolated that can bypass the need for SEC4, reflecting a role for Sec1 downstream of Sec4. The interaction of Sec1 with other Sec proteins will be tested. Overexpression of SEC9 can suppress a mutation in the effector region of Sec4, but not elsewhere, suggesting an interaction with this important domain. This will be probed biochemically and genetically. The role of the novel myosin, Myo2, in exocytotic and endocytic transport will be examined. We will determine if the predicted Cdc2 phosphorylation site in Myo2 is used in cell cycle regulation of its function.